KR20120088021A - Novel compounds with antioxidant activity and/or lipid peroxidation inhibitory activity and isolation method of the compounds from salicornia herbacea l - Google Patents

Abstract

PURPOSE: A novel compound with antixodiation or lipid peroxidation inhibition is provided to be applied to a functional food and pharmaceutical composition. CONSTITUTION: A novel compound of isoquercitrin 6"-O-methyloxalate is denoted by chemical formula 1. A novel compound of methyl 4-carffeoyl-3-dihydrocaffeoyl quinate(saliconate) is denoted by chemical formula 2. The novel compound is isolated from Salicornia herbacea L. A method for preparing the novel compound comprises: a step of preparing Salicornia herbacea L. extract; a step of concentrating the extract at reduced pressure; a step of fractioning the concentrate by solvent fraction to prepare a butanol fraction; and a step of purifying the butanol fraction by Amberlite XAD-2 column chromatography and HPLC using the reverse phase column.

Description

Novel compounds with antioxidant activity and / or lipid peroxidation inhibitory activity and isolation method of the compounds from Salicornia herbacea L.}

The present invention relates to a novel compound having at least one of antioxidant activity and peroxide production inhibitory activity, a compound separation method capable of separating the new compound from Salicornia herbacea L. and the use of the new compound.

Salicornia herbacea L. is a plant belonging to the family of the Mingaceae, which grows on the seashore of the saltwaters of the west coast and Ulleungdo. It is a plump, protruding grass in Korean. Stalks of stalks are segmented and prominent, with branches branched two or three times. Eggplants are fleshy, large, dark green, without leaves. It grows about 10 ~ 40 cm in height, and the flower blooms as if it appears green at the end of the branch from June to August, and the fruit ripens flat and black in October. Stalks are green from spring to summer, and dark red in autumn.

Although there is no record of using stalk as a herb or food in Korea, people who live by the sea eat it with herbs. It is also said that in the private sector, when you feel sick and have no food in the spring, eating herbs in the mouth will clear your eyes and improve the taste of rice. It is said to have been written. In Europe, young stems are often eaten as salads, and in Japan, they are designated as natural monuments.

Recently in Korea, the unique taste and various physiological effects of tongung bark is being orally edible in traditional methods such as fresh herbs in Haenam, Jeollanam-do, and are sold in the form of living or powder. In particular, it has been reported that blood cholesterol lowering effect, blood total lipid content and triglyceride content reduction, arteriosclerosis, hyperlipidemia, fatty liver prevention effect, weight gain inhibition, etc. (Park Si-Hyung et al. , J. Korean Soc. Appl. Biol) Chem ., 47, 120-123, 2004), but the research on the causative agent is very poor.

Meanwhile, the compounds isolated so far have been isolated from isoramnetin 3- O -beta-di-glucopyranoside, quasicitin 3-o-beta- Quercetin 3- O- β-D-glucopyranoside, 3-cafeoyl-4-dihydrocafeoylquinic acid (tongmadic acid) (3-caffeoyl-4- dihydrocaffeoyl quinic acid; tungtungmadic acid ), Beta-sitosterol (β-sitosterol), stigmasterol (uracil) has been reported. Among these, isoramnetine 3-o-beta-di-glucopyranoside has been reported to have antioxidant and antidiabetic effects, and quasicitin 3-o-beta-di-glucopyranoside has been reported to have antioxidant effects. In addition, 3-caffeoyl-4-dihydrocafeoylquinic acid (Tungmadic acid) has been reported to improve cirrhosis in liver cirrhosis-induced mice (YC Chung et al., J. Toxicol. Pub. Health , 22, 267-273, 2006).

In addition, various studies have been reported on the immunomodulatory function, antioxidative effect, serum lipid improvement effect, and applicability of functional cosmetics (Jin-tae Lee et al . , Kor. J. Herbology , 17, 61-69, 2002; Kyung Ran Kim et al., J. Korean Soc.Food Sci.Nutr ., 37, 170-176, 2008). However, the study of the ingredients included in the barb is not enough.

  The present inventors have completed the present invention by searching for and isolating substances contained in the bark, focusing on antioxidant compounds known to be involved in various disease prevention.

Therefore, an object of the present invention is isoquarcitrin 6 '' -O- methyloxalate and methyl 4-caffeoyl-3-, which are novel compounds having antioxidant activity and anti-oxidant inhibitory activity. Dihydrocafeoylquinate (salconate) [methyl 4-caffeoyl-3- dihydrocaffeoyl quinate (salicornate)].

Another object of the present invention is to identify a variety of antioxidants in the bark bark, and to provide a novel antioxidant compound separation method that can separate the new compound having antioxidant activity and peroxidation inhibitory activity from the bark barb and confirm the structure.

Still another object of the present invention is to provide a functional food and pharmaceutical composition having at least one of antioxidant activity and inhibitory activity for inhibiting peroxidation, including a novel antioxidant compound, which has been separated and structured from the bark, as an active ingredient.

The objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a new compound named isoquarcitrin 6 '' -O- methyloxalate having the formula (1).

[Formula 1]

The present invention also provides a novel compound named methyl 4-cafeoyl-3-dihydrocafeoylquinate (salconate) {methyl 4-caffeoyl-3-dihydrocaffeoyl quinate (salicornate) having the following formula (2): do.

[Formula 2]

In addition, the present invention comprises the steps of obtaining a tung bark extract; Concentrating the knurled bark extract under reduced pressure; Obtaining a butanol fraction through a solvent fraction of the concentrate; And purifying the butanol fraction by HPLC using Amberlite XAD-2 column chromatography and reversed phase column.

In a preferred embodiment, the step of obtaining the spongy extract is extracted by slicing the ground portion of the spongy, adding methanol and homogenizing.

In addition, the present invention includes any one or more of isoquacitrin 6 ''-o-methyloxalate or methyl 4-cafeoyl-3-dihydrocafeoyl quinate as an active ingredient during the antioxidant activity and the inhibitory activity of peroxide production It provides a functional food characterized in that it has one or more.

The present invention also provides a pharmaceutical composition for antioxidants comprising any one or more of isoquacitrine 6 ''-o-methyloxalate or methyl 4-cafeoyl-3-dihydrocafeoylquinate as an active ingredient.

The present invention also provides a pharmaceutical composition for inhibiting the formation of peroxides comprising any one or more of isoquacitrine 6 ''-o-methyloxalate or methyl 4-cafeoyl-3-dihydrocafeoylquinate as an active ingredient. do.

The present invention has the following excellent effects.

First, according to the present invention, isoquacitrin 6 '' -O- methyloxalate and methyl 4-cafeoyl-3-di, which are novel compounds having antioxidant activity and inhibitory activity for peroxide production Hydrocafeoylquinate (salconate) [methyl 4-caffeoyl-3- dihydrocaffeoyl quinate (salicornate)] may be provided.

In addition, according to the present invention, it is possible to provide a novel antioxidant compound separation method that confirms the presence of various antioxidant substances in the bark node, and isolates a new compound having antioxidant activity and peroxidation inhibitory activity from the bark node and confirms its structure. .

According to the present invention, a functional food and pharmaceutical composition having one or more of an antioxidant activity and a peroxide production inhibitory activity can be provided by including a novel antioxidant compound separated and structured from the stalk as an active ingredient.

1 is a separation, purification process diagram of the novel compound 1 and novel compound 2 obtained in Examples 1 and 2 of the present invention,
FIG. 2 is a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity evaluation graph of the fractions after solvent fractionation of the Rhizome extract obtained during the separation and purification of FIG.
3 is a gHMBC correlation (arrow) and structure diagram of isoquacitrin 6 '' -O -methyloxalate, a novel compound 1 ;
Figure 4 is a novel compound 4-methyl 2-dihydro-3-cafe five days five days cafe quinolyl carbonate (live Rico carbonate; compound 2) [methyl 4-caffeoyl- 3-dihydrocaffeoyl quinate (salicornate)] of gHMBC correlation (arrow) And schematic,
5 is a graph showing the DPPH radical-scavenging activity of the novel compounds 1 and 2 isolated from spurs;
6 is a graph showing the inhibitory effect of cholesteryl ester hydroperoxide (CE-OOH) production in rat plasma of Novel Compounds 1 and 2 isolated from spurs;

Although the terms used in the present invention have been selected as general terms that are widely used at present, there are some terms selected arbitrarily by the applicant in a specific case. In this case, the meaning described or used in the detailed description part of the invention The meaning must be grasped.

Hereinafter, with reference to the accompanying drawings and preferred embodiments will be described in detail the technical configuration of the present invention.

However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Like reference numerals used to describe the present invention throughout the specification denote like elements.

Technical features of the present invention are confirmed by the TLC-DPPH method (Takao T. et al., Biosci . Biotechnol . Biochem ., 58, 1780-1783, 1994) that there are various antioxidants on the stalks, Two novel compounds that exhibit at least one of antioxidant activity and peroxide production inhibitory activity were isolated and their structure determined.

The two novel compounds isolated in the present invention are isoquarcitrin 6 '' -O- methyloxalate and methyl 4-cafeoyl-3-dihydrocafeoylquinate ( Saliconate) [methyl 4-caffeoyl-3- dihydrocaffeoyl quinate (salicornate)].

First, isoquacitrin 6 '' -O- methyloxalate in the novel compound of the present invention has the following Chemical Formula 1.

 [Formula 1]

In addition, methyl 4-cafeoyl-3-dihydrocafeoylquinate (saliconate) [methyl 4-caffeoyl-3-dihydrocaffeoyl quinate (salicornate)] of the novel compounds of the present invention has the following formula (2).

[Formula 2]

Specifically, as will be described later, the novel compounds of the present invention have excellent antioxidant and / or peroxide production inhibitory activity that is expected to be derived from the catechol backbone possessed by these compounds.

Example 1

1. Extract of Tung Bark and Solvent Fraction

Salicornia herbacea L. was prepared by harvesting 15-20 cm of raw grass from Yawol-ri, Hwangsan-myeon, Yeongsan-myeon, Yeonggwang-gun, Jeollanam-do, Korea. Homogenized with (BM-2 Nissei boi-mixer, Nehonseiki kaiseiki LTD., Japan) and extracted for 18 hours at room temperature and dark conditions. After performing suction filtration (No. 2, Whatman), 10 L of methanol was added to the obtained residue, and it extracted and filtered again by the same method. The extracted filtrate was concentrated under reduced pressure at 39 ° C using a vacuum concentrator (A-3S, Eyela, Tokyo, Japan) equipped with an aspirator (CCA-1110, Eyela, Tokyo, Japan). The water suspension (3 L) of the obtained concentrate (417.33 g) was solvent-separated in sequence by repeating 3 L of hexane, ethyl acetate, and saturated butanol three times each.

2. Isolation of Compound 1 Designated as Isoquacitrin 6 ''-O-Methyloxalate

As shown in FIG. 1, Amberlite XAD-2 column chromatography was performed on 33.94 g of a butanol layer obtained by solvent fractionation of the methanol extract of Tung Krabi. The sample was concentrated, dissolved in H ₂ O adjusted to pH 2.65 with trifluoroacetic acid (TFA), and filled with a column (4.5) filled with XAD-2 (1 L, 521 μm, SUPELCO, USA), equivalent to about 30 times the sample. ㅧ 80 cm). Elution of 1.8 L of 20, 40, 60, 80 and 100% MeOH content (v / v) in H ₂ O adjusted to pH 2.65 with TFA and H ₂ O, followed by 2 L of acetone I was. The 50 fractions obtained were grouped by TLC-DPPH analysis to determine the distribution trend of antioxidant active compounds, and then the antioxidants were extracted from BH (60% MeOH, 2.21 g) with relatively high antioxidant activity. Gradient between 35% methanol solution adjusted to pH 2.65 with TFA and 50% methanol using a prep-ODS column [shim-pack-prep-ODS (H) Kit 20 ㅧ 250 mm] to isolate (gradient, 55 min) HPLC was performed by applying the elution method (10.0 ml / min). As a result, a total of eight groups were obtained from BH1 to 8, among which BH5 ( t _R 31.5 min, 16.8 mg) having high antioxidant activity was subjected to HPLC under the same conditions as above, and had a peak of t _R 37.9 min. A single compound (Compound 1 , 3.8 mg, yellow powder) was isolated and named isoquacitrin 6 ''-o-methyloxalate.

Example 2

The BH fraction was fractionated by HPLC, followed by another fraction BH6 ( t _R 32.3 min, 22.4 mg) having high antioxidant activity other than fraction BH5 (the fraction from which New Compound 1 was isolated). The same procedure was followed to isolate a single compound (compound 2 , 4.5 mg, white powder) with a peak of t _R 43.6 min and with methyl 4-cafeoyl-3-dihydrocafeoylquinate (salconate). Named it.

Experimental Example 1

TLC analysis and antioxidant activity of each fraction after solvent fractionation of the Rheumulus extract were confirmed by the following experiment.

First, antioxidant activity was assayed for each fraction obtained by the solvent fraction of Example 1 using the TLC-DPPH method. That is, after the methanol extract, the hexane layer, the ethyl acetate layer, the butanol layer and the water layer were prepared in the same amount of equivalent amount (equivalent to 20 mg of fresh weight of the bark), and then spotted on TLC, butanol / acetic acid / water = 4: 1: 1 (v / v) using a solvent system, and then sprayed with a 1,1-diphenyl-2-picrylhydrazyl (DPPH) ethanol solution to determine the white-bleached portion as an antioxidant activity positive (TLC-DPPH method; Takao T. et al., Biosci . Biotechnol . Biochem ., 58, 1780-1783, 1994) was used to compare the tendency of the antioxidants contained in each layer.

As a result, spots of various antioxidant compounds were detected in the ethyl acetate layer, hexane layer, butanol layer and water layer, and reacted with various concentrations of samples and DPPH radicals of constant concentration (250 μM, final concentration). Relative radical-scavenging activity (SC ₅₀ ) was compared and reviewed, and the results are shown in FIG. 2.

As shown in Figure 2, the activity of the ethyl acetate layer and butanol layer was the highest. The water layer showed very low activity, and the hexane layer contained (a large amount) of a compound that absorbs light at 517 nm, resulting in a decrease in activity as the amount of sample added increased. It was supposed to be.

On the other hand, the ethyl acetate layer showed the highest antioxidant activity, but due to the presence of too many active compounds, it was determined that the purification and isolation process would be difficult and time-consuming. However, the butanol layer was somewhat less than the acetylacetate layer, but showed an antioxidant activity comparable to that of the ethyl acetate layer. As a result, it turns out that a butanol layer is suitable for refine | purifying and isolation of an antioxidant compound.

Experimental Example 2

Structural Analysis of Compound 1

From the HR-ESI-MS (postive) spectrum of compound 1, the molecular weight peak m / z 573.0855 [M + Na] ⁺ was observed, suggesting that the compound's molecular formula is C ₂₄ H ₂₂ O ₁₅ ( m / z 550). . And five sp ² carbon proton siganls [δ 6.21 (1H, br. S, H-6), 6.41 (1H, 5), attributed to the quercustin, the flavonoid from the ¹ H-NMR (600 MHz, CD ₃ OD) spectrum. br.s, H-8), 7.62 (1H, d, J = 2.4 Hz, H-2 ′), 6.75 (1H, d, J = 8.7 Hz, H-5 ′) and 7.58 (1H, dd, J = 8.7, 2.4 Hz, H-6 ')] and 7 types of sp ³ belonging to 1 type of glucose carbon proton signals (δ 5.17 (1H, d, J = 8.0 Hz, H-1 "), 3.38-3.47 (4H, m, H-2"-5 "), 4.26 (1H, dd, J = 12.0, 1.8 Hz, H-6a ″) and 4.11 (1H, dd, J = 12.0, 5.4 Hz, H-6b ″)], and one methoxyl proton signal was observed at δ 3.68 (3H, s).

^A total of 24 carbon signals were observed from ¹³ C-NMR, including 15 aglycone carbon signals [carbonyl carbon signal {δ 179.6 (C-4)}, 14 sp ² carbon signals (δ 166.2-94.9)], six monosaccharide derived carbon signals [anomer carbon signal (δ 104.4), 5 non-anomer carbon signals (δ 78.0-65.2)] and two carbonyl carbon signals [ δ 168.1 (C-1 '''), δ 168.9 (C-2''')] and one methoxyl carbon signal [δ 52.9 (-OCH ₃ )], respectively.

For more accurate structural analysis, 2D-NMR analysis of gHSQC, gHMBC, etc. was performed. As a result, gHMBC showed an ester bond with δ 168.1 (C-1 ′ '') of methyloxalate from δ 4.26 (H-6 ″ a) proton and δ 3.68 (-OCH ₃ Correlation between the carbon signals of methyloxalate (δ 168.9, C-2 ''') was observed from the proton signals, and their binding patterns were confirmed.

Compound 1 was determined to be isoquacitrin 6 '' -O- methyloxalate from Table 1, in which the results of this instrument analysis are shown, and it was found to be a new compound.

Table 1 shows ¹ H- (600 MHz) and ¹³ C-NMR (150 MHz) data of isoquarcitrin 6 '' -O -methyloxalate (Compound 1 ) (CD ₃ OD , TMS).

In addition, gHMBC correlation (arrow) and structure of isoquacitrin 6 '' -O -methyloxalate are shown in FIG. 3.

Carbon δ _H (int, mult., J in Hz) δ _C 2 - 159.6 3 - 135.5 4 - 179.6 5 - 163.2 6 6.21 (1 H, s) 100.1 7 - 166.2 8 6.41 (1 H, s) 94.9 9 - 158.7 10 - 105.8 One' - 123.3 2 ㅄ 7.62 (1 H, d, 2.4) 117.6 3 ㅄ - 146.1 4 ㅄ - 149.9 5 ㅄ 6.75 (1H, d, 8.7) 116.1 6 ㅄ 7.58 (1H, doublet of 2.4, 8.7) 123.5 One 5.17 (1H, d, 7.8) 104.4 2 3.47 (1H, d, 6.5, 8.0) 75.8 3˝ 3.42 (1H, d, 7.0, 8.0) 78.0 4 3.30 (1H, d, 7.5, 8.0) 71.3 5˝ 3.38 (1 H, m) 75.6 6a˝ 4.26 (1H, doublet of 1.8, 12.0) 65.2 6b˝ 4.11 (1H, doublet of doublets, 5.4, 12.0) One?? - 168.1 2?? - 168.9 -OCH ₃ 3.68 (3H, s) 52.9

Experimental Example 3

Structural Analysis of Compound 2

Compound 2 was found to have a molecular weight peak of m / z 531.1508 [M-H] ⁺ in HR-ESI-MS (negative) analysis as a white powder, and the molecular formula of this compound was C ₂₆ H ₂₈ O ₁₂ ( m / z 532). ¹ methoxyl group signal (δ 3.74, 3H, s) from the ¹ H-NMR (600 MHz, CD ₃ OD) spectrum, 9 sp ³ carbon proton signals [δ 2.15 (1H, m, H-2a), 2.08 (1H, m, H-2b), 5.49 (1H, m, H-3), 5.01 (1H, dd, J = 8.7, 3.3 Hz, H-4), 4.27 (1H, m, H-5) , 2.24 (1H, m, H-6a), 2.07 (1H, m, H-6b), 2.71 (2H, J = 7.2, 7.5, 7.8 Hz, t, H-7 Hz) and 2.49 (2H, m, H-8 ㅄ)], 8 sp ² carbon proton signals [δ 6.59 (1H, d, J = 2.0 Hz, H-2 ㅄ), 6.62 (1H, d, J = 8.0 Hz, H-5 ㅄ) , 6.65 (1H, dd, J = 8.0, 2.0 Hz, H-6 Hz), 7.05 (1H, d, J = 1.8 Hz, H-2 Hz), 6.78 (1H, d, J = 8.4 Hz, H- 5 Hz), 6.96 (1H, dd, J = 8.4, 1.8 Hz, H-6 Hz), 7.60 (1H, d, J = 15.6 Hz, H-7 Hz) and 6.29 (1H, d, J = 15.6 Hz) , H-8 '). From these data, the presence of two tri-substituted benzene rings, a trans-type olefinic double bond, a -CH ₂ -CH ₂ -substructure and quinic acid, respectively, was suggested.

A total of 26 carbon signals were observed from ¹³ C-NMR, including three carboxylic carbon signals [δ 175.3 (C-7), 173.8 (C-9 ') and 168.5 (C-9'). 1 methoxyl carbon signal [δ 53.2 (-OCH ₃ )], 4 methylene carbon signals [δ 39.1 (C-2), 38.4 (C-6), 31.5 (C-7 ㅄ), 37.5 (C- 8 ㅄ)], three methine carbon signals [δ 69.1 (C-3), 75.4 (C-4), 69.0 (C-5)], and eight double bond carbon signals [δ 116.5 (C-2 ㅄ). ), 116.5 (C-5 ㅄ), 120.6 (C-6 ㅄ), 115.3 (C-2 ˝), 116.6 (C-5 ˝), 123.3 (C-6 ˝), 147.8 (C-7 ˝), 114.8 (C-8 ')], and seven quaternary carbon signals [δ 76.2 (C-1), 133.4 (C-1'), 146.3 (C-3 '), 144.8 (C-4'), 127.8 (C-1 ′), 146.9 (C-3 ′), 149.9 (C-4 ′).

Based on the above data, it was thought that quinic acid had ester linkage with caffeic acid, dihydrocaffeic acid and methyl group. 2D-NMR analysis of gHSQC, gCOSY, gHMBC, etc. was performed for more accurate structural analysis. As a result, C-7 (δ 175.3) of quinic acid from δ 3.74 (-OCH ₃ ) protons and dihydrocaffeic acid from δ 5.49 (H-3) protons were determined in the gHMBC spectrum. Correlation between C-9 ′ (δ 173.8) and C-9 ″ (δ 173.8) of caffeic acid from δ 5.01 (H-4) protons was observed, respectively. From this result, it was confirmed that the methyl group at the seventh position of the compound quinic acid, the dihydrocaffeic acid at the third position, and the caffeic acid at the fourth position were ester-bonded, respectively.

Compound 2 was determined to be methyl 4-cafeoyl-3-dihydrocafeoylquinate (salconate) [methyl 4-caffeoyl-3-dihydrocaffeoyl quinate (salicornate)] from the results of this instrumental analysis shown in Table 2. It turned out to be a compound.

Table 2 shows ¹ H- (600 MHz) and ¹³ C of methyl 4-cafeoyl-3-dihydrocafeoylquinate (saliconate; compound 2 ) [methyl 4-caffeoyl-3-dihydrocaffeoyl quinate (salicornate)] -NMR (150 MHz) data (CD ₃ OD, TMS).

In addition, gHMBC correlation (arrow) and structure of methyl 4-cafeoyl-3-dihydrocafeoylquinate (salconate; compound 2 ) [methyl 4-caffeoyl-3-dihydrocaffeoyl quinate (salicornate)] are shown in FIG. As shown in.

Carbon δ _H (int, mult., J in Hz) δ _C One - 76.2 2a 2.15 (1 H, m) 39.1 2b 2.08 (1 H, m) 3 5.49 (1H, m) 69.1 4 5.01 (1H, doublet of 3.3, 8.7) 75.4 5 4.27 (1H, m) 69.0 6a 2.24 (1 H, m) 38.4 6b 2.07 (1 H, m) 7 - 175.3 One' - 133.4 2 ㅄ 6.59 (1H, d, 1.8) 116.5 3 ㅄ - 146.3 4 ㅄ - 144.8 5 ㅄ 6.62 (1H, d, 7.8) 116.5 6 ㅄ 6.65 (1H, dd, 2.1, 8.1) 120.6 7 ㅄ 2.71 (2H, t, 7.2, 7.5, 7.8) 31.5 8 ㅄ 2.49 (2H, m) 37.5 9 ㅄ - 173.8 One - 127.8 2 7.05 (1H, d, 1.8) 115.3 3˝ - 146.9 4 - 149.9 5˝ 6.78 (1 H, d, 8.4) 116.6 6˝ 6.96 (1H, dd, 1.8, 8.4) 123.3 7˝ 7.60 (1 H, d, 15.6) 147.8 8 6.29 (1H, d, 15.6) 114.8 9˝ - 168.5 -OCH ₃ 3.74 (3H, s) 53.2

Experimental Example 4

DPPH radical-scavenging activity test

1,1-diphenyl-2-picrylhydrazyl (DPPH, Wako, Japan) ethanol solution was prepared so that the final concentrations of the compounds 1 and 2 isolated from the bark obtained in Examples 1 and 2 were 10 μM. The final concentration of (50 μL) was set to 250 μM to prepare a solution such that the final volume was 200 μL. After the mixed solution was reacted for 20 minutes in the dark at room temperature, the absorbance was measured at 517 nm (Versa max microplate reader, Molecular Devices, USA), and the results are shown in FIG. 5. Chlorogenic acid was used as a control. The values given are standard deviations of the results obtained from three replicates. The compound 1 is 1 in FIG. 2 is the compound 2-chloro-CA represents a transgenic Acid (chlorogenic acid), a, b are each other means that there is significant difference (p <0.05) between the group injeongdoem indicated by different symbols.

In the activity evaluation, Compound 2 showed lower tendency than Compound 1 , but no significant difference was observed. Compounds 1 and 2 showed significantly higher activity than chlorogenic acid used as a control.

Experimental Example 5

Inhibitory Activity of Cholesteryl Ester Hydroperoxide (CE-OOH) Production in Rat Plasma

1) Isolation of experimental animals and plasma

The mice used in the experiment were 6-week-old Sprague-Dawley, 180-200 g body wt. Males (Samtako, Osan-si, Gyeonggi-do) were bred in plastic cages for 3 days. Growth conditions were 20 ㅁ 2 ℃, humidity was 50-60%, light-dark cycle was maintained every 12 hours, and the diet and water were freely ingested and purified in the laboratory environment. Fasting 15 hours before the experiment, 3 hours before the water, and then opened under ether anesthesia, and collected by a heparin-added syringe from the aorta and centrifuged (3000 rpm, 4 ℃, 20 minutes, VS-15 CFN, VISION, KOREA) ), And the obtained upper layer (plasma, plasma) was taken and stored frozen at -40 ℃ until just before use.

2) Oxidation and Peroxide (CE-OOH) Analysis in Rat Plasma

640 μL of PBS buffer (pH 7.4) was added to 250 μL of mouse plasma, and 10 nmol (final concentration of 10 μM) of Compounds 1 and 2 obtained in Examples 1 and 2 were dissolved in PBS buffer, respectively. Oxidation was initiated by adding 100 μL of PBS buffer solution so that the copper ion (CuSO ₄ ) finally reached 100 μM. The mixed solution was shaken incubated at 37 ° C. for 7 hours, and 100 μL of 3 mL of methanol and n -hexane containing 2.5 mM BHT were added and mixed in vortex. The supernatant was taken up in a concentration vessel, and 3 mL of n -hexane was added to the lower layer solution and mixed again with vortex. The supernatant was taken, mixed with the n -hexane layer of the previous step, concentrated, and the obtained concentrate was dissolved in 100 μL of a methanol / trichloromethane (95: 5, v / v) solution, and the concentration of CE-OOH in the sample was determined. UV detector (280) with a flow rate of 1.0 mL / min (model 510 solvent delivery system, Waters, USA) using 97% methanol as mobile phase using a TSKgel ODS-80Ts column (4.6 mm 250 mm, Tosoh, Japan) nm, SPD 10A, SHIMADZU, Japan) was used for HPLC, and the results are shown in FIG. This experiment also used chlorogenic acid (chlorogenic acid) as a control. In FIG. 6, C represents an additive-free group, 1 represents Compound 1 , 2 represents Compound 2 , and CA represents a chlorogenic acid addition group.

As shown in the results of the activity evaluation in Figure 6 did not show a significant difference in activity between the compounds 1 and 2 , the addition of antioxidant-free (C) and chlorogenic acid without the addition of antioxidant compounds to rat plasma solution When comparing (CA), a 5-fold or more CE-OOH production inhibitory effect was observed. That is, Compound 2 showed lower tendency than Compound 1 , but no significant difference was observed, and Compound 1 and 2 showed significantly higher activity than chlorogenic acid used as a control.

These results showed that compounds 1 and 2 exhibited radical scavenging in solution as well as inhibitory effect of plasma peroxide generation induced by copper ions.

Thus, compounds 1 (isoquacitrine 6 ''-o-methyloxalate) and compound 2 (methyl 4-cafeoyl-3-dihydrocafeoylquinate) of the present invention are antioxidative and / or peroxide inhibitory activity As described above, when the food is prepared by including any one or more of Compound 1 or Compound 2 of the present invention, a functional food having antioxidant activity and / or peroxide production inhibitory activity may be prepared.

In addition, Compound 1 (Isoquacitrin 6 ''-O-Methyloxalate) and Compound 2 (Methyl 4-cafeoyl-3-dihydrocafeoylquinate) or nontoxic salts thereof of the present invention are used in pharmaceutical or veterinary medicine. Pharmaceutical compositions comprising an acceptable liquid or solid carrier can be provided. Here, the pharmaceutical composition may be used for the use of an antioxidant or a peroxide generating inhibitor, and a pharmaceutically or veterinary acceptable liquid or solid carrier is not limited, and all known ones may be used, and thus the detailed description thereof will be omitted.

The present invention has been shown and described with reference to the preferred embodiments as described above, but is not limited to the above embodiments, those skilled in the art without departing from the spirit of the present invention. Various changes and modifications will be possible.

Claims (7)

A novel compound named isoquarcitrin 6 '' -O- methyloxalate having the formula 1 below.
[Formula 1]

A novel compound named methyl 4-cafeoyl-3-dihydrocafeoylquinate (salconate) having the formula (II) {methyl 4-caffeoyl-3-dihydrocaffeoyl quinate (salicornate)}.
(2)

Obtaining a bark extract;
Concentrating the knurled bark extract under reduced pressure;
Obtaining a butanol fraction through a solvent fraction of the concentrate; And
The butanol fraction is purified by HPLC using Amberlite XAD-2 column chromatography and reversed phase column separation method according to claim 1 or 2 of the novel compound.
The method of claim 3, wherein
The step of obtaining the rung bark extract is characterized in that the new compound separation method characterized in that the extracted by adding the methanol and homogenized after cutting the ground portion of the rung bark. Antioxidant activity and peroxide inhibitory activity comprising any one or more of isoquacitrin 6 ''-o-methyloxalate of claim 1 or methyl 4-cafeoyl-3-dihydrocafeoylquinate of claim 2 as an active ingredient Functional food characterized in that it has one or more of.
A pharmaceutical composition for antioxidants comprising any one or more of isoquacitrin 6 ''-o-methyloxalate of claim 1 or methyl 4-cafeoyl-3-dihydrocafeoylquinate of claim 2 as an active ingredient.
A pharmaceutical composition for use in inhibiting peroxide production comprising at least one of isoquacitrine 6 ''-o-methyloxalate of claim 1 or methyl 4-cafeoyl-3-dihydrocafeoylquinate of claim 2 as an active ingredient.

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